Resveratrol compounds, specially trans-resveratrol (3,5,4′-trihydroxystilbene), presents antioxidant, anti-inflammatory, antiviral, cardioprotective, neuroprotective, and chemoprotective activities. Besides protecting against infections and ischemia, they reduce obesity and prevent aging. Trans-resveratrol is a polyphenol found mainly in red grape skin and red wine. The more intense wine or grape color, the bigger their polyphenol content is. Studies seem to indicate that trans-resveratrol can lower levels of low-density lipoproteins, also known as LDL cholesterol or “bad” cholesterol, and increase levels of high-density lipoproteins, HDL cholesterol, or “good” cholesterol. Especially when in oxidized state, LDL can be retained in blood vessel walls resulting in the formation of atheroma plaques. These plaques cause atherosclerosis, which leads to blood vessel obstruction. Trans-resveratrol stimulates HDL production by liver, decreases LDL production and avoids circulating LDL oxidation; therefore, it plays a role in reducing the risk of cardiovascular disorders, such as myocardial infarct.
Trans-resveratrol is also widely known as a modulator of the expression and activity of a protein class called sirtuin. Sirtuin modulators are compounds that up regulate (activating or stimulating), down regulate (inhibiting or eliminating), or change the functional property or the biological activity of sirtuin protein.
Sirtuin is a member of the family of protein deacetylases (Sirtuins), or rather of the Sir2 family, which includes yeast Sir2 protein (GenBank Accession No. P53685), C. elegans Sir-2.1 (GenBank Accession No NP—501912), and human SIRT1 (GenBank Accession No. NM—012238 and NP—036370 or AF083106) and SIRT2 (GenBank Accession No NM—012237, NM—030593, NP—036369, NP13 085096, and AF083107).
Sirtuin protein modulators, such as resveratrol compounds, particularly trans-resveratrol derivatives, actuate to minimize or treat aging diseases, chronic degenerative diseases of circulatory and neurological systems, such as ocular diseases (WO 2006/127987), psychopathologies (WO 2006/138418), diabetes (WO 2006/104586), cancer (WO 2006/102557), and obesity (US 2006/111435).
However, trans-resveratrol has low water solubility, less than 0.03 g/L, which complicates its oral administration (bioavailability) and its use as additive in non-alcoholic beverages. To bypass this restriction, without the synthesis of another derivative, is used in the present invention the preparation, under specific conditions, of a complex of resveratrol compound, preferably trans-resveratrol, with a cyclodextrin, preferably beta-cyclodextrin (β-cyclodextrin), presenting substantial and surprising increase in solubility.
Cyclodextrins are cyclic oligosaccharides, also known as cycloglucans or Schardinger dextrins, produced from starch by enzymatic action. Cyclodextrins can be natural or semi-synthetic. Natural cyclodextrins are, for example, α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin. The use of natural cyclodextrins as a carrier of active principles has been considered restrict due to its relatively low solubility in water, particularly when it comes to β-cyclodextrin (Uekama, K.; Hirayama, F. and Irie, T. “Cyclodextrin Drug Carrier Systems”. Chem. Rev. 98(5), 2045-2076, 1998). To bypass such limitation, the semi-synthetic derivatives of β-cyclodextrin prepared by methylation or hydroxy-alkylation of the hydroxyl groups of β-cyclodextrin were obtained, resulting in amorphous cyclodextrins that are more water soluble.
Beta-cyclodextrin (β-CD) is a crystalline cyclodextrin composed of seven units of D(+)-glucopyranoses linked α-(1-4), being the most employed in food area. As for its metabolism, it is considered digestible especially in large intestine when it is fermented by bacterial flora, fact determined in animal and human experiments. β-CD is not toxic neither genotoxic, even when ingested in high concentrations (Diniz, A. C. P. et al. Cienc. Tecnol. Aliment. 2005. 25(2), 197).
Recently, it was reported that aqueous solutions of trans-resveratrol and β-CD, in a ratio of 1:1, increase resveratrol water solubility, as well as its bioavailability in model systems (López-Nicolás J. M, et al J. Chromatogr. A, 2006, 158; Lucas-Abellán, C. Food Chem., 2007, 39 and Lucas-Abellán, C.; Fortea, I., López-Nicolás, J. M. and Núñez-Delicado, E. “Cyclodextrins as resveratrol carrier system”. Food Chemistry, 104 (2007) 39-44, available online in Jan. 4, 2007). However, said document does not reveal the process for obtaining β-CD/trans-resveratrol (β-CD/Res) chemical complex, neither how obtaining it to use in formulations. In addition, the document does not specify the alleged “solubility increase”, but it does mention the great influence of complexation conditions in β-cyclodextrin/resveratrol complex properties.
Bertacche et al. (Bertacche, V., Lorenzi, N., Nava, D., Pini, E. and Sinico, C. “Host-Guest Interaction Study of Resveratrol with Natural and Modified Cyclodextrins”. Journal of Inclusion Phenomena and Macrocyclic Chemistry, Vol. 55 (3-4). August 2006, 279-287) revealed the use of cyclodextrin to form cyclodextrin/resveratrol complex, mentioning natural cyclodextrins (α, β or γ) or modified ones (resulting from any of these forms). However, there is no specification about relative concentrations of any of such cyclodextrins in relation to resveratrol; neither there is formation of a cyclodextrin-resveratrol crystalline complex with amazingly high solubility.
It can be concluded from scientific literature that the formation of a complex with cyclodextrin is equivalent to a reaction, that is, different parameters or process conditions result in products with different characteristics. This can be observed, for example, in the several different approaches of patent literature, described below.
Patent literature considers some documents related to processes for obtaining trans-resveratrol, highlighting some of them that cite the use of cyclodextrin. Although none of the documents discloses or even indirectly suggests the inventive concept of the present invention, some of them are mentioned below as reference.
Some patent applications show the use of cyclodextrins and trans-resveratrol as a part of pharmaceutical formulations obtained by a physical mixture with other substances, but the isolation of trans-resveratrol/β-cyclodextrin (β-CD/Res) complex does not happen.
International patent application WO 2007/009997, filed by Actimex S.r.l. (Italy) and entitled “Composition Containing Micronutrients with Improved Anti-oxidant Activity and Use Thereof”, presents compositions in fine powder form having ternary mixtures. Such ternary composition has an active component, a carrier and an auxiliary co-grinding agent, since the process of obtainment include grinding for at least 90 minutes. Although in said document resveratrol is mentioned as one of the active components and cyclodextrin as one of the possible carriers, the obtainment of a β-CD/Res chemical complex with high water solubility is not shown.
United States patent application US 2006/111318, filed by Advanced Medicine Research Institute and entitled “Agent for treating eye diseases”, shows an agent for treating eye diseases containing sexual steroid hormone and a sirtuin modulator, which can be resveratrol. In said document, the process for preparing eye drops includes water dissolution of resveratrol, forming a suspension, and the addition of γ-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin. The increase in solubility by complexation with this cyclodextrins mixture is attributed to the use of amorphous cyclodextrins instead of crystalline cyclodextrins, such as β-cyclodextrin.
Japanese patent application JP 2000/344622, filed by Sunstar Inc. and entitled “Stabilization of Stilbenic Compound and Plant Extract Containing the same, and Food, Medicine, Cosmetic or Oral Cavity Preparation Stably Compounded with Stilbenic Compound and Plant Extract Containing the same”, shows the combination of stilbenic compounds with cyclodextrin, as a way to increase stability and efficiency of stilbenic compound absorption. Although resveratrol is one of the possible stilbenic compounds in said document, the obtainment of a crystalline chemical complex of resveratrol with cyclodextrin compound with high water solubility is not revealed.
Other patent applications show the obtainment of cyclodextrin complexes with other molecules than trans-resveratrol, which, in such documents, appears only as a part of the formulation. For example, the international patent application WO 2003/077860 shows the cyclodextrin complex with 3-β-acetyl-11-keto-β-boswellic acid; WO 2004/087121, provides the obtainment of cyclodextrin complex with digitalis glycosides; and WO 2006/083458 relates to the preparation of cyclodextrin complex with diindolylmethane.
The complexation of poorly soluble compounds by cyclodextrins, in which the molecules of said compounds are “guests” in the cavity of cyclodextrin molecule, is carried out in solid medium as well as liquid medium or even in semisolid medium. Such methods have advantages and disadvantages that depend on several factors and may be cited as the most important the steric hindrance, the stability of said compounds, the presence of non-reacted components, and the crystallinity of chosen cyclodextrin.
A representative example of the difficulties to be overcome in complexation process is the obtainment of inclusion complex of piroxicam (a practically insoluble substance)-cyclodextrin. The methods known by the state of the technique include reactions that can happen in solid as well as liquid medium and even in semisolid medium.
Concerning to the complexation of piroxicam in liquid medium: (1) in EP 153998, the complexation of piroxicam with a cyclodextrin selected from α-, β-, and γ-cyclodextrin group is described. The reaction can be carried out by: (a) direct dissolution of piroxicam in an aqueous solution of selected cyclodextrin and separation of the complex by crystallization; (b) piroxicam dissolution in an organic solvent and mixture of the resulting organic solution, under stirring, with an aqueous solution of selected cyclodextrin and separation of the complex by crystallization; (c) dissolution of piroxicam and cyclodextrin compounds, under stirring, in a water-ammonia solution and separation of the complex by drying; and (d) dissolution of piroxicam and cyclodextrin compounds, under stirring, in a water-hot ammonia solution and separation of the complex by freeze-drying or atomization in air flow; (2) in WO 03105906, a complexation process is described: piroxicam and β-cyclodextrin are diluted in water in the presence of ammonium hydroxide under a temperature of 60° C., the solution being then cooled to nearly −10° C. and, afterwards, lowering the temperature of the frozen solution to at least −20° C., preferably from −30° C. to −40° C.; finally, the solution is dried under vacuum; and (3) in WO 06013039, a preparation process of a piroxicam:β-cyclodextrin (1:2.5) inclusion compound is described piroxicam and β-cyclodextrin are diluted, in a molecular ratio of 1:2.5, in hot water and in the presence of ammonium hydroxide; the resulting solution undergoes drying by spray-drying with rigorous control of input and output temperatures of the drying gas and posterior separation of the complex into powder. These different methods for obtaining the same complex (piroxicam/β-cyclodextrin) show how complicate its preparation is and aim to solve problems related to scale enlargement, from laboratory to industry, and for obtaining an amorphous type of piroxicam/β-cyclodextrin complex to improve solubility and reduce the risks of crystallization of the complex during storage.
Concerning the complexation of piroxicam in semisolid medium, document WO 03053475 presents a preparation process of inclusion complexes of an active principle (in example 1, piroxicam) and a cyclodextrin (α-, β-, γ-cyclodextrin or a semisynthetic cyclodextrin, such as hydroxypropyl-β-cyclodextrin) by mixing said active principle and cyclodextrin, both in the form of finely divided powders, in the presence of small amounts of water or alcoholic, acid or base aqueous solutions. The resulting mixture is treated in a microwave oven and the resulting product is dried under vacuum at room temperature or with heating at a temperature under 50° C. It is mentioned that the technique improvement to obtain cyclodextrin complexes relates to the obtainment of complexes that are more wettable, have a greater solubility equilibrium and a faster dissolution in aqueous medium comparing to the crystalline active principle, resulting in more auspicious pharmacokinetics and, therefore, best therapeutic results.
Finally, concerning the complexation of piroxicam in solid medium, document EP 449167 presents a preparation process of piroxicam-cyclodextrin complexes, where both compounds, in the form of finely divided powders, are mixed in solid state and grinded under high-energy conditions in a steam chamber. The resulting product is dried under vacuum and sifted to eliminate any aggregates. It is mentioned that the improvement aimed at obtaining a piroxicam-cyclodextrin complex with high density and big surface area, properties that, when combined with an extremely thin size of the particle, result in an appropriate product to pharmaceutical compositions of oral, rectal, and topical administration.
One of the methods suggested in the state of the technique to improve the stability of “guest” molecules in cyclodextrin complexes is described in WO 06137959. The method shown in said document comprises the mixture of cyclodextrin and an emulsifier, the addition to this mixture of the compound to be complexed, diluted in an appropriate solvent, to the formation of the aimed complex, and finally the addition of non-complexed cyclodextrin to the inclusion complex of cyclodextrin to form a stabilizing system of the “guest” molecule.
The teachings of WO/2005/111224 are useful to complexation technique of insoluble or slightly water soluble substances with cyclodextrins. In the examples of said document, a process for obtaining a coenzyme Q10 and β-cyclodextrin soluble in water is described. The dissolution of β-cyclodextrin in water is carried out at a temperature of 30° C., preferably between 55° C. and the boiling temperature. Coenzyme Q10 is added either in solid form or dissolved in an appropriate solvent, preferably in solid form. The stirring is carried out at increased temperature, then at room temperature for several hours. Afterwards, the complex is isolated by filtration, decantation, or water evaporation. Optionally the complex is dried.
U.S. Pat. No. 6,884,885 describes a process for preparing active principle and cyclodextrins complexes to increase the yield of the complexes. The process comprises the dissolution of cyclodextrin and guest molecule in a liquid solvent at a temperature ranging from 20° C. to 100° C., preferably from 60° C. to 80° C. Cyclodextrin should be at a concentration of about 15% (p/p) or above, and said solution should have a molecular ratio of cyclodextrin to said molecule of about 1:1 to about 10:1. Then the mixture of the solution is carried out to allow a complex to form as a precipitate. The precipitate (complex) is separated.
The teachings of the documents mentioned above show the difficulties of the complexation of insoluble or slightly water soluble active principles by cyclodextrins. There are many parameters to be controlled in the obtainment of water-soluble complexes presenting purity level appropriate to pharmaceutical or nutraceutical use, acceptable industrial yield and stability of the complex during storage of the products having them. It should be noted that several documents about the state of the art mention the fact that the presence of non-reacted raw material in the final complex can cause stability problems of the products during storage.
Although cyclodextrins can be considered interesting carriers to active principles, especially to hydrophobes, in pharmaceutical formulations the selection of appropriate cyclodextrin to a specific active principle and determination of reaction conditions to the preparation of the aimed complex are fundamental. The literature has some important contributions that help us in this significant task. For example, the advantages and physico-chemical characteristics of cyclodextrins are detailed in the work of Uekama and collaborators (1998), where it is mentioned that hydroxyalkylated cyclodextrins increase the solubility of complexes formed with hydrophobic substances due to their amorphous feature (Part III, Item A.1 (“Hydroxyalkylated Cyclodextrins”)). On the other hand, Uekama et al. also show that amorphous forms are easily transformed into stable crystalline form during handling and storage of medicines, being important the control of the crystallization, of the polymorphic transition and the formation of whiskers (flexible and strong crystals), especially in correlation with cyclodextrin effect in the physical stability of active principles in solid state. Despite Uekama and co-workers remarking the differentiated performance of hydroxypropyl-β-cyclodextrin, this behavior is not the same for all the active principles, for example, when β-cyclodextrin is indicated to the production of isosorbide-5-mononitrate pills (vasodilator) to avoid whiskers (part III, Item B.2 (“In the Solid State”).
Chinese patent application CN 2005/1566054, filed by Institute of Materia Medica (China) and entitled “Resveratrol oligo cattail compounds, its manufacturing process, pharmaceutical combination, and uses thereof”, shows the obtainment of a cyclodextrin complex with cis-ε-viniferine, an oligomeric stilbenic compound of Veratrum album.
International patent application WO 2004/103265, filed by Enprani Co. and entitled “Whitening and Antioxidative Cosmetic Composition Containing Resveratrol and Method for Preparing the Same”, describes the use of cyclodextrin (hydroxypropyl-β-cyclodextrin) and polyethyleneglycol to stabilize a cosmetic composition having trans-resveratrol. In said composition, another stabilizer is also used, selected from alpha-lipoic acid, Phellodendron extract or Alteromonas ferment extract.
Chinese patent application CN 1500479, published on Jun. 2, 2004 and entitled “Resveratrol, Piceid and its Derivative and its Preparation”, describes the obtainment of hydroxypropyl-β-cyclodextrin clathrates in the ratio of 1:50. The preparation process described in said document comprises resveratrol dissolution in an organic solvent 1-5 times, hydroxypropyl-beta-cyclodextrin dissolution in distilled water 5-50 times, drip of this aqueous solution in the suspension with resveratrol, mixture agitation, filtration and freeze drying to obtain the product in the form of clathrate which is intended for injection use.
In the International patent applications WO 2006/127987 (corresponding to US 2006/0292099) and WO 2006/105403, whose applicant is Sirtris Pharmaceuticals, Inc. and entitled “Treatment of Eye Disorders with Sirtuin Modulators”, ophthalmic preparations comprising inclusion complexes formed by resveratrol and cyclodextrins are described. It is mentioned that, preferably, cyclodextrins are modified to increase resveratrol solubility and, hence, its bioavailability (pages 148-150 and 159-162 from documents WO 2006/105403 and WO 2006/127987, respectively). In such documents, it was suggested the use of amorphous cyclodextrin, with a new approach to obtain the complex and surprising results in terms of increased solubility and its magnitude. It is important to note that emphasis is given to the fact that the use of cyclodextrin derivatives in amorphous form is advantageous, such as hydroxypropyl-β-cyclodextrin, because non-modified cyclodextrins (α-, β- and γ-cyclodextrin) tend to crystallize, being, therefore, less water soluble than the amorphous one. However, the disadvantages resulting from the handling of amorphous substances in pharmaceutical techniques are not mentioned, which may render some pharmaceutical forms not feasible, such as, for example, solid pharmaceutical forms such as pills. This is definitely a limiting factor in the preparation of pharmaceutical compositions, such limitation being circumvented when preparing pharmaceutical compositions with crystalline complex of resveratrol and cyclodextrin compound forms—which is one of the technical features of the present invention.
Therefore, no document found in prior art showed a concrete report or even suggestion that crystalline CD/Res complex would provides a substantial increase in resveratrol solubility and, hence, resveratrol enhanced bioavailability and, at the same time, keeping complex stability during final product handling and storage. The substantial solubility increase of the complex of the present invention, compared to the solubility of other resveratrol and cyclodextrin complexes known in the art, is surprising and circumvent several technical limitations of the currently known approaches.